Are there functional P2X receptors on cell bodies in intact dorsal root ganglia of rats?

P2X purinoceptors have been suggested to participate in transduction of painful stimuli in nociceptive neurons. In the current experiments, ATP (1-10 mM), alpha,beta-methylene-ATP (10-30 mu M) and capsaicin (10 nM-1 mu M) were applied to neurons impaled with high resistance microelectrodes in rat dorsal root ganglia (L4 and L5) isolated in vitro together with the sciatic nerve and dorsal roots. The agonists were either bath applied or focally applied using a picospritzer. GABA (100 mu M) and 40-80 mM K+ solutions gave brisk responses when applied by either technique. Only three of 22 neurons with slowly conducting axons (C cells) showed evidence of P2X-purinoceptor-mediated responses. Only two of 13 cells which responded to capsaicin (putative nociceptors), and none of 29 cells with rapidly conducting axons (A cells), responded to the purinergic agonists. When acutely dissociated dorsal root ganglion cells were studied using patch-clamp techniques, all but four of 30 cells of all sizes responded with an inward current to either ATP or alpha,beta-methylene-ATP (both 100 mu M). Our data suggest that few sensory cell bodies in intact dorsal root ganglia express functional purinoceptors. (C) 1998 IBRO. Published by Elsevier Science Ltd.

The eyes of deep-sea fish II. Functional morphology of the retina

Three different aspects of the morphological organisation of deep-sea fish retinae are reviewed: First, questions of general cell biological relevance are addressed with respect to the development and proliferation patterns of photoreceptors, and problems associated with the growth of multibank retinae, and with outer segment renewal are discussed in situations where there is no direct contact between the retinal pigment epithelium and the tips of rod outer segments. The second part deals with the neural portion of the deep-sea fish retina. Cell densities are greatly reduced, yet neurohistochemistry demonstrates that all major neurotransmitters and neuropeptides found in other vertebrate retinae are also present in deep-sea fish. Quantitatively, convergence rates in unspecialised parts of the retina are similar to those in nocturnal mammals. The differentiation of horizontal cells makes it unlikely that species with more than a single visual pigment are capable of colour vision. In the third part. the diversity of deep-sea fish retinae is highlighted. Based on the topography of ganglion cells, species are identified with areae or foveae located in various parts of the retina, giving them a greatly improved spatial resolving power in specific parts of their visual fields. The highest degree of specialisation is found in tubular eyes. This is demonstrated in a case study of the scopelarchid retina, where as many as seven regions with different degrees of differentiation can be distinguished, ranging from an area giganto cellularis, regions with grouped rods to retinal diverticulum. (C) 1998 Elsevier Science Ltd. All rights reserved.

Retinal and lenticular ultrastructure in the aestivating salamanderfish, Lepidogalaxias salamandroides (Galaxiidae, Teleostei) with special reference to a new type of photoreceptor mosaic

The salamanderfish, Lepidogalaxias salamandroides (Galaxiidae, Teleostei) is endemic to southwestern Australia and inhabits shallow, freshwater pools which evaporate during the hot summer months. Burrowing into the substrate in response to falling water levels allows these fish to aestivate for extended periods of time while encapsulated in a mucous cocoon even when the pools contain no water. Only a few minutes after a major rainfall, these fish emerge into relatively clear water which subsequently becomes laden with tannin, turning the water black and reducing the pH to approximately 4.3. As part of a large study of the visual adaptations of this unique species, the retinal and lenticular morphology of the aestivating salamanderfish is examined at the level of the light and electron microscopes. The inner retina is highly vascularised by a complex system of vitreal blood vessels, while the outer retina receives a blood supply by diffusion from a choriocapillaris. This increased retinal blood supply may be an adaptation for reducing the oxygen tension during critical periods of aestivation. Large numbers of Muller cells traverse the thickness of the retina from the inner to the outer limiting membranes. The ganglion cells are arranged in two ill-defined layers, separated from a thick inner nuclear layer containing two layers of horizontal cells by a soma-free inner plexiform layer. The photoreceptors can be divided into three types typical of many early actinopterygian representatives; equal double cones, small single cones and large rods (2:1:1). These photoreceptors are arranged into a unique regular square mosaic comprising a large rod bordered by four equal double cones with a small single cone located at the corner of each repeating unit. The double cones may optimise perception of mobile prey which it tracks by flexion of its head and neck and the large rods may increase sensitivity in the dark tannin-rich waters in which it lives. Each single cone also possesses a dense collection of polysomes and glycogen (a paraboloid) beneath its ellipsoid, the first such finding in teleosts. The retinal pigment epithelium possesses melanosomes, pha,oocytes and a large number of mitochondria. The anatomy of the retina and the photoreceptor mosaic is discussed in relation to the primitive phylogeny of this species and its unique life history.

The ocular morphology of the southern hemisphere lamprey Geotria australis gray with special reference to optical specialisations and the characterisation and phylogeny of photoreceptor types

This paper describes the ocular morphology of young adults of the southern hemisphere lamprey Geotria australis, the sole representative of the Geotriidae, and makes comparisons with those of holarctic lampreys (Petromyzontidae). As previously reported for the holarctic lamprey Ichthyomyzon unicuspis [Collin and Fritzsch, 1993], the lens of G. australis is non-spherical and possesses a cone-shaped posterior that may be capable of mediating variable focus. The avascular retina of G. australis is well differentiated, containing three retinal ganglion cell populations, three layers of horizontal cells and three photoreceptor types, in contrast to petromyzontids that contain only two photoreceptor types (short and long), G. australis possesses one rod-like (R1) and two cone-like (C1 and C2) photoreceptors. Although the rodlike receptor in G. australis may be homologous with the short receptors of holarctic lampreys, the two cone-like receptors have morphological characteristics that differ markedly from those of the long receptors of their holarctic counterparts. The features which distinguish the two cone-like receptors from those of the long receptor type in holarctic lampreys are the characteristics of the mitochondria and the presence of large amounts of two different types of stored secretory material in the endoplasmic reticulum of the myoid (refractile bodies). The endoplasmic reticulum of each receptor type has a different shape and staining profile and is polymorphic, each showing a continuum of distension. It is proposed that the presence of two cone-like photoreceptors with different characteristics would increase the spectral range of G. australis and thus be of value during the parasitic phase, when this lamprey lives in the surface marine waters. The irideal flap, present in G. australis but not petromyzontids, would assist in reducing intraocular flare during life in surface waters. The results of this study, which are discussed in the context of the proposed evolution of lampreys, emphasise that it is important to take into account the characteristics of the eyes of southern hemisphere lampreys when making generalizations about the eyes of lampreys as a whole.

Tubular eyes of deep-sea fishes: A comparative study of retinal topography

The world's deep oceans are home to a number of teleosts with asymmetrical or tubular eyes. These immobile eyes possess large spherical lenses and subtend a large binocular visual field directed either dorsally or rostrally. Derived from a lateral non-tubular eye, the tubular eye is comprised of a thick main retina, subserving the rostrally or dorsally directed binocular visual field, and a thin accessory retina subserving, the lateral, monocular visual field. The main retina is thought to receive a focussed image, while the accessory retina is too close to the lens for a focussed image to be received. Several species also possess retinal diverticula, which are small evaginations of differentiated retina located in the rostrolateral wall of the eye and thought to increase the visual field. In order to investigate the spatial resolving power of these retinae (main, accessory and diverticulum), the distribution of cells within the ganglion cell layer was analysed from retinal wholemounts and sectioned material in ten species representing four genera. In all species, the main retina possesses a marked increase in cell density towards a specialised retinal region (area centralis), with a centro-peripheral gradient range between 7.1 and 60:1 and a peak density range of between 30 and 55 x 10(3) cells per mm(2). The accessory retinae and the transitional zone between the main and accessory retinae possess relatively low cell densities (between 1 and 10 x 10(3) cells per mm(2)) and lack an area centralis. Retinal diverticula examined in four species possess mean ganglion cell densities of between 7.2 and 109.4 x 10(3) cells per mm(2). Analyses of soma areas show that the ganglion cell layer of most species possesses cells with areas in a range of 8.0 to 15.4 mu m(2) in the main retina and between 15.1 and 17.4 mu m(2) in the accessory retina. The peak spatial resolving power of the main retina of the ten species varies from 4.1 to 9.1 cycles per degree. The positions of the retinal areae centrales relative to each species' binocular visual field are discussed in relation to what is known of feeding behaviour of these fishes in the deep-sea.

Retinal neurons: cell types and coupled networks

Retinal neurons with distinct dendritic morphologies are likely to comprise different cell types, subject to three important caveats. First, it is necessary to avoid creating “artificial” cell types based on arbitrary criteria—for example, the presence of two or three primary dendrites. Second, it is essential to take into account changes in morphology with retinal eccentricity and cell density. Third, the retina contains imperfections like any natural system and a significant number of retinal neurons display aberrant morphologies or make aberrant connections that are not typical of the population as a whole. Many types of retinal ganglion cells show diverse patterns of tracer coupling, with the simplest pattern represented by the homologous coupling shown by On-Off direction-selective (DS) ganglion cells in the rabbit retina. Neighboring DS ganglion cells with a common preferred direction have regularly spaced somata and territorial dendritic fields, whereas DS ganglion cells with different preferred directions may have closely spaced somata and overlapping dendritic fields.

Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions

Lipins constitute a novel family of Mg2+-dependent phosphatidate phosphatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differentially expressed among tissues. Compatible with organism tissue complexity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) measured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addition, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concentrated in the internal rim of the ring canal. These specific subcellular localizations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family.

PRECLINICAL INVESTIGATION OF THE RETINAL BIOCOMPATIBILITY OF SIX NOVEL VITAL DYES FOR CHROMOVITRECTOMY

Purpose: To investigate the retinal biocompatibility of six novel vital dyes for chromovitrectomy. Methods: An amount of 0.05 mL of 0.5% and 0.05% light green (LG), fast green (FG), Evans blue (EB), brilliant blue (BriB), bromophenol blue (BroB), or indigo carmine (IC) was injected intravitreally in the right eye, whereas in the left eye balanced salt solution was applied for control in rabbits` eyes. Clinical examination, fluorescein angiography, histology with light microscopy, and transmission electron microscopy were performed after 1 and 7 days. Retinal cell layers were evaluated for morphologic alterations and number of cells. The electroretinographic changes were assessed at baseline, 24 hours and 7 days. Results: Fluorescein angiography disclosed hypofluorescent spots only in the 0.5% EB group. Light microscopy and transmission electron microscopy disclosed slight focal morphologic changes in eyes exposed to 0.05% IC, FG, BriB, similar to the control at 1 and 7 days. In the lower dose groups, EB, LG, and BroB caused substantial retinal alterations by light microscopy. At the higher dose, BroB and EB produced diffuse cellular edema and vacuolization within the ganglion cells, bipolar cells, and photoreceptors. FG and IC at 0.5% caused slight retinal alterations similar to balanced salt solution injection. LG at 0.5% caused diffuse vacuolization of bipolar cells after 1 and 7 days. Injection of 0.5% EB caused a significant decrease in neuroretinal cell counts in comparison to control eyes in the 7-day examination (P < 0.05). Electroretinography revealed intermittent prolonged latency and decreased amplitude in eyes injected with 0.5% EB, LG, BriB, and BroB, while at the lower dose, only LG and EB induced few functional changes. Conclusion: The progressive order of retinal biocompatibility, from safest to most toxic, was IC, FG, BriB, BroB, LG, EB.

Localization of neurotransmitters and calcium binding proteins to neurons of salamander and mudpuppy retinas

We wished to identify the different types of retinal neurons on the basis of their content of neuroactive substances in both larval tiger salamander and mudpuppy retinas, favored species for electrophysiological investigation. Sections and wholemounts of retinas were labeled by immunocytochemical methods to demonstrate three calcium binding protein species and the common neurotransmitters, glycine, GABA and acetylcholine. Double immunostained sections and single labeled wholemount retinas were examined by confocal microscopy. Immunostaining patterns appeared to be the same in salamander and mudpuppy. Double and single cones, horizontal cells, some amacrine cells and ganglion cells were strongly calbindin-immunoreactive (IR). Calbindin-IR horizontal cells colocalized GABA. Many bipolar cells, horizontal cells, some amacrine cells and ganglion cells were strongly calretinin-IR. One type of horizontal cell and an infrequently occurring amacrine cell were parvalbumin-IR. Acetylcholine as visualized by ChAT-immunoreactivity was seen in a mirror-symmetric pair of amacrine cells that colocalized GABA and glycine. Glycine and GABA colocalized with calretinin, calbindin and occasionally with parvalbumin in amacrine cells. (C) 2001 Elsevier Science Ltd. All rights reserved.

Differential perturbation of neuronal and glial glutamate transport systems in retinal ischaemia

Glutamate is the major excitatory neurotransmitter in the retina and is removed from the extracellular space by an energy-dependent process involving neuronal and glial cell transporters. The radial glial Muller cells express the glutamate transporter, GLAST, and preferentially accumulate glutamate. However, during an ischaemic episode, extracellular glutamate concentrations may rise to excitotoxic levels. Is this catastrophic rise in extracellular glutamate due to a failure of GLAST? Using immunocytochemistry, we monitored the transport of the glutamate transporter substrate, D-aspartate, in the retina under normal and ischaemic conditions. Two models of compromised retinal perfusion were compared: (1) Anaesthetised rats had their carotid arteries occluded for 7 days to produce a chronic reduction in retinal blood flow. Retinal function was assessed by electroretinography. D-aspartate was injected into the eye for 45 min, Following euthanasia, the retina was processed for D-aspartate. GLAST and glutamate immunocytochemistry. Although reduced retinal perfusion suppresses the electroretinogram b-wave, neither retinal histology, GLAST expression, nor the ability of Muller cells to uptake D-aspartate is affected. As this insult does not appear to cause excitotoxic neuronal damage, these data suggest that GLAST function and glutamate clearance are maintained during periods of reduced retinal perfusion. (2) Occlusion of the central retinal artery for 60 min abolishes retinal perfusion, inducing histological damage and electroretinogram suppression. Although GLAST expression appears to be normal. its ability to transport D-aspartate into Muller cells is greatly reduced. Interestingly, D-aspartate is transported into neuronal cells, i.e. photoreceptors, bipolar and ganglion cells. This suggests that while GLAST is vitally important for the clearance of excess extracellular glutamate, its capability to sustain inward transport is particularly susceptible to an acute ischaemic attack. Manipulation of GLAST function could alleviate the degeneration and blindness that result from ischaemic retinal disease. (C) 2001 Elsevier Science Ltd, All rights reserved.

Roles of fibroblast growth factors in the inner ear

The basic biology of the fibroblast growth factor (FGF) receptors and their splice variants is first reviewed, followed by a review of the known roles of FGFs in the inner ear. They include induction of the otocyst by FGF19, followed by FGF3 in further development of the otocyst. In later development, FGF3 or FGF10 acting on FGF receptor 2b is likely to be involved in development of the walls of the cochlear spaces, while FGF receptor 3 is involved in differentiation of the pillar cells of the organ of Corti. FGF1 and FGF2 act as trophic factors for the developing cochlear nerve fibres. Copyright (C) 2002 S. Karger AG, Basel.

Distribution of two splice variants of the glutamate transporter GLT-1 in the developing rat retina

The distributions of a carboxyl terminal splice variant of the glutamate transporter GLT-1, referred to as GLT-1B, and the carboxyl terminus of the originally described variant of GLT-1, referred to hereafter as GLT-1alpha, were examined using specific antisera. GLT-1B was present in the retina at very early developmental stages. Labelling was demonstrable at embryonic day 14, and strong labelling was evident by embryonic day 18. Such labelling was initially restricted to populations of cone photoreceptors, the processes of which extended through the entire thickness of the retina and appeared to make contact with the retinal ganglion cells. During postnatal development the GLT-1B-positive photoreceptor processes retracted to form the outer plexiform layer, and around postnatal day 7, GLT-1B-immunoreactive bipolar cells appeared. The pattern of labelling of bipolar cell processes within the inner plexiform layer changed during postnatal development. Two strata of strongly immunoreactive terminals were initially evident in the inner plexiform layer, but by adulthood these two bands were no longer evident and labelling was restricted to the somata and processes (but not synaptic terminals) of the bipolar cells, as well as the somata, processes, and terminals of cone photoreceptors. By contrast, GLT-1alpha appeared late in postnatal development and was restricted mainly to a population of amacrine cells, although transient labelling was also associated with punctate elements in the outer plexiform layer, which may represent photoreceptor terminals, (C) 2002 Wiley-Liss, Inc.

Direction selectivity in the retina

The neuronal circuitry underlying the generation of direction selectivity in the retina has remained elusive for almost 40 years. Recent studies indicate that direction selectivity may be established within the radial dendrites of 'starburst' amacrine cells and that retinal ganglion cells may acquire their direction selectivity by the appropriate weighting of excitatory and inhibitory inputs from starburst dendrites pointing in different directions. If so, this would require unexpected complexity and subtlety in the synaptic connectivity of these CNS neurons.

Diverse synaptic mechanisms generate direction selectivity in the rabbit retina

The synaptic conductance of the On-Off direction-selective ganglion cells was measured during visual stimulation to determine whether the direction selectivity is a property of the circuitry presynaptic to the ganglion cells or is generated by postsynaptic interaction of excitatory and inhibitory inputs. Three synaptic asymmetries were identified that contribute to the generation of direction-selective responses: (1) a presynaptic mechanism producing stronger excitation in the preferred direction, (2) a presynaptic mechanism producing stronger inhibition in the opposite direction, and (3) postsynaptic interaction of excitation with spatially offset inhibition. Although the on- and off-responses showed the same directional tuning, the off-response was generated by all three mechanisms, whereas the on- response was generated primarily by the two presynaptic mechanisms. The results indicate that, within a single neuron, different strategies are used within distinct dendritic arbors to accomplish the same neural computation.